Periodontitis is a polymicrobial disease caused by the coordinated action of a complex microbial community, which results in inflammation of tissues that support the teeth. Periodontitis is a widespread and serious health condition that occurs in moderate form in 30% to 50% of American adults and in severe form in 10% of adults. The goal of this research program is to identify in situ the molecular mechanisms of pathogenesis by which periodontitis progress in some sites while in others the disease remains latent. Metagenomic and metatranscriptomic analysis (gene expression analysis of entire complex bacterial communities in situ) provides the information required to understand the activity and relative importance of the constituents in the pathogenic biofilm during periodontal infection. To this end we propose the following Specific Aims: Aim 1. Determine differences in prevalence and relative abundance of species in microbial communities from progressing and non-progressing periodontal sites. Aim 2. Identify in situ genes that are differentially expressed in microbial communities from progressing and non-progressing periodontal sites. We have successfully applied metatranscriptomic techniques to the study of environmental samples. The key step of the method to assess expression analysis in whole microbial communities is the linear amplification of small quantities of RNA extracted from the bacterial assemblages. The final amplified RNA is a direct representation of the relative abundance of mRNA in the original sample. The second aspect that facilitates metatranscriptomic analysis of whole bacterial communities is that pyrosequencing techniques allow now to sequence large amounts of DNA avoiding cloning biases associated with classical techniques. The target subject population will consist of 20 chronic periodontitis individuals. This subject population was chosen because chronic periodontitis is the most commonly encountered type of periodontitis and thus, the microbial changes observed will be relevant to a large proportion of subjects with periodontal disease. The patients will be followed monthly for a period of 6 months, during which they will undergo clinical monitoring to determine which samples will be used for compassion of progressing and non-progressing sites by metagenomic and metatranscriptomic analysis. Identification of critical genes that are required for pathogenesis and information about their differential expression can be used to develop novel targeted approaches to early-stage diagnosis, treatment, monitoring and prevention. Moreover, the potential impact extends beyond the study of periodontitis because the same principles and methods potentially can be applied to other polymicrobial diseases. We believe that the team we have assembled for this project (Forsyth Institute with help of Broad Institute and the Department of Biostatistics at Harvard School of Public Health) has all the qualifications to accomplish successfully the goals proposed in the present application. PUBLIC HEALTH RELEVANCE: Periodontitis is a polymicrobial disease caused by complex microbial community, which could result in the loss of teeth. The goal of this project is to understand which organisms are active and what they are doing during periodontitis progression to develop specific treatments against them.